Micropropagation

  Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.

Micropropagation is used to multiply novel plants, such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well to vegetative propagation.

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Methods

Establishment

  Micropropagation begins with the selection of plant material to be propagated, clean stock material for propagation is important in the production of the healthiest plants. Often plants are first virus indexed to make sure they are clean and free of viruses. Once the plant material is chosen for culture, the collection of explant(s) begins and is dependent of the type of tissue to be used, and can include stem tips, anthers, petals, pollen and others. The explant material is then sterilized on their surfaces, usually in multiple courses of bleach and alcohol washes and finally rinsed in sterilized water. This small portion of plant tissue, which may be as small as a cell, is placed on a growth medium, typically containing sucrose as an energy source and one or more plant growth regulators (plant hormones). Usually the medium is thickened with agar to create a gel which supports the explant during growth. Some plants are easy to grow on simple mediums but other plants need more complicated mediums before they will successively grow; some mediums include vitamins, minerals and amino acids. The medium is sterilized during preparation to prevent fungal and bacterial contaminations that can outgrow and smother the explant. Autoclaves and filter sterilization are used to remove potential contaminates, under smaller scales of production a pressure cooker is often used.

The plant tissue should now begin to grow and differentiate into new tissues. For example, media containing cytokinins are used to create branched shoots from plant buds.

Multiplication

Multiplication is the taking of tissue samples produced during the first stage and increasing there number. Following the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds or thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and mediums. If the plant material grown is callus tissue, it can be placed in a blender and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue. If the tissue is grown as small plants called plantlets, hormones are often added that cause the plantlets to produce many small offshoots that can be removed and recultured.

Pretransplant

This stage involves treating the plantlets/shoots produced to encourage root growth and "hardening." It is performed in vitro, or in a sterile "test tube" environment.

Root growth does not always occur in the earlier stages in plant cell culture, and is of course a requirement for successful plant growth after the micropropagation procedure. It is often performed in vitro by transferring the plantlets to a growth medium containing auxin(s) which stimulate root initiation. The pretransplant stage is not always performed; Some plants are micropropagated and grown in culture and normal cuttings are made that are then rooted ex vitro.

"Hardening" refers to the preparation of the plants for a natural growth environment. Until this stage, the plantlets have been grown in "ideal" conditions, designed to encourage rapid growth. Due to lack of necessity, the plants are likely to be highly susceptible to disease and often do not have fully functional dermal coverings and will be inefficient in their use of water and energy. In vitro conditions are high in humidity and plants grown under these condition do not form a working cuticle and stomata that keep the plant from drying out, when taken out of culture the plantlets need time to adjust to more natural environmental conditions. Hardening typically involves slowly weaning the plantlets from a high-humidity, low light, warm environment to what would be considered a normal growth environment for the species in question. This is done by moving the plants to a location high in humidity, such as a green house with regular mist watering.

Transfer from culture

In the final stage of plant micropropagation, the plantlets are removed from the plant media and transferred to soil or (more commonly) potting compost for continued growth by conventional methods.

This stage is often combined with the "pretransplant" stage.

Advantages

Micropropagation has a number of advantages over traditional plant propagation techniques:

• The main advantage of Micropropgation is the production of many plants that are clones of each other.
• Micropropagation can be used to produces disease-free plants.
• Micropropagation produces rooted plantlets ready for growth, and saves time for the grower, instead of seeds or cuttings.
• It has an extraordinarily high fecundity, producing thousands of propagules in the same time it would take a conventional technique to produce tens or hundreds
• It is the only viable method of regenerating genetically modified cells or cells after protoplast fusion.
• It is a good way of multiplying plants which produce seeds in uneconomical amounts (if at all) or whose seed can't be stored (vgr. recalcitrant seeds).
• Micropropagation often produces more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods.

Disadvantages

Micropropagation may appear to be the perfect means of multiplying plants, but it has associated problems:

• It is very expensive, and can have a labour cost of more than 70%
• An infected plant sample can produce infected progeny. This is uncommon if the stock plants are carefully screened and vetted to prevent culturing plants infected with virus or fungus.
• Not all plants can be successfully tissue cultured, often because the proper medium for growth is not know or the plants produce secondary metabolic chemicals that stunt or kill the explant.
• Sometimes plants or cultivars do not come true to type after being tissue cultured, this is often dependent on the type of explant material utilized during the initiation phase or the result of the age of the cell or propagule line.
• Some plants are very difficult to disinfest of fungal organisms.

The greatest limitation is the cost of production, and for most plants seeds, which are normally disease free and produced in good numbers readily produce plants in good numbers at a lower cost. For this reason, many plant breeders do not utilize micropropagation because of the prohibitive cost, other breeders use it to produce stock plants that are then used for seed multiplication.

Mechanisation of the process would eliminate most of the labour cost associated, but this has proven difficult so far despite active attempts to develop this technology.

See also

• Bioreactor
• Floriculture
• Fragmentation (biology)
• Murashige and Skoog medium
• Plant propagation
• Plant tissue culture
• Vegetative reproduction

References